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Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles
Neurochemistry International 34 (1999) 509±516
Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles Teresa Iantomasi, Fabio Favilli, Maria T. Vincenzini* Department of Biochemical Sciences, University of Firenze, viale Morgagni 50, Firenze, 50134 Italy Received 1 October 1998; received in revised form 1 February 1999; accepted 1 February 1999
Abstract Glutathione (GSH) transport was studied in synaptosomal membrane vesicles (SMV) of rat cerebral cortex. The present study shows that GSH uptake into SMV occurs very quickly in a time-dependent manner into an osmotically active intravesicular space. The initial rate of transport followed Michealis-Menten saturation kinetics with a Km 4.5 2 0.8 mM that shows a high anity of the transporter for GSH. Therefore GSH uptake in SMV occurs by a mediated transport system which can be activated by either an inward gradient of cations, like Na+ or K+, or membrane depolarization. These results, together with those obtained by valinomycin-induced K+ diusion potential, indicate that GSH synaptosomal transport is electrogenic by a negative charge transfer. The increase of GSH uptake measured by trans-stimulation experiments con®rms a GSH bidirectional mediated transport which seems susceptible of modulation by changes in ionic ¯uxes and in the membrane potential. These results may indicate a possible involvement of this transporter in the role suggested for GSH in synaptic neurotransmission; also considering that GSH precursor of neuroactive aminoacids (glyeine, glutamate), may contribute to regulate their level in synapses. Finally, a GSH transporter in synaptosomes may contribute to maintaining the GSH homeostasis in cerebral cortex, where decreases of GSH levels have been related to susceptibility to neuropathologies. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Glutathione transporter; Rat; Synaptosomes membranes
1. Introduction GSH is a major and ubiquitous intracellular antioxidant (Ishikawa and Sies, 1989). GSH concentrations in the brain of dierent animals are high, ranging from 0.5 to 3 mM (Orlowsky and Karkowsky, 1976; Sagara et al., 1996). GSH has been found prevalently in primary astrocyte culture (Huang and Philbert, 1995; Sagara et al., 1996; Juurlink et al., 1996) while little GSH is present in neurones (Raps et al., 1989) and some studies have also demonstrated a compartmentaAbbreviations: SMV, synaptosomal membrane vesicles; g-GT, gglutamyltranspeptidase; AT125, Acivicin (LaS; 5 S)-a-amino-3chloro4,5-dihydro-5-isoxozoleacetic acid). * Corresponding author. Tel.: +39-55-416686; fax: +39-554222725. E-mail address: [email protected]®.it (M.T. Vincenzini)
lisation of GSH and GSH-related enzymes within neurones with GSH accumulation in axon terminals in synaptosomal fraction (Pileblad et al., 1991; Favilli et al., 1994). GSH antioxidant and detoxi®cating action in brain tissue and particularly in synaptosomes has been established (Pileblad et al., 1989; Cooper and Kristal, 1997), together with pathological changes of GSH metabolism associated with neurodegenerative processes of the brain (Perry and Yong, 1987; Mizui et al., 1992; Bains and Shaw, 1997). However, the speci®c physiological role of GSH in the brain still remains to be explained. Several recent studies have suggested a possible additional role for GSH as a neuromodulator and/or neurotransmitter (Guo et al., 1992; Lanius et al., 1994; Ogita et al., 1995). In fact, there is evidence that GSH may in¯uence neurotransmission by interacting directly with the speci®c binding site of glutamic acid on the synaptic membranes and/or as precursor
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of neurotransmitter aminoacids (Ogita et al., 1995). GSH-speci®c binding sites in brain synaptic membranes and in cultured astrocytes have been found (Ogita and Yoneda, 1989; Guo et al., 1992). Moreover, it has been demonstrated that both GSH and cystine are released into perfusates from rat brain slices on stimulation by K+ in a Ca2+ dependent manner (ZaÈngerle et al., 1992), and astrocytes are also known to eux GSH at a high rate (Yudko et al., 1990; Sagara et al., 1996; Dringen et al., 1997). Previous data have also demonstrated GSH transport in rat blood-brain barrier and in perfused guinea pig brain (Kannan et al., 1990; Zlokovic et al., 1994), and recently three distinct GSH transporter activities have been identi®ed in bovine brain capillary (Kannan et al., 1996). Many data indicate that synaptosomes contain a heterogeneous population of transporters of aminoacids and their precursors and that uptake processes are in some cases the primary mechanism for the neurotransmitters remotion from the synapse (Dowd et al., 1996). In view of these previous ®ndings, we investigated whether GSH can cross SMV of rat cerebral cortex by mediated transport in order to modulate cerebral GSH turnover, levels of neuroactive aminoacids (L-glutamate, glycine and L-cysteine) and to regulate mechanisms of neurotransmission. 2. Experimental procedures [Glycine-2,3H] GSH (spec. act. 43.8 Ci/mmol) and acid glutamic, L-[14C(U)] (spec. act. 281.4 mCi/mmol) were obtained from New England Nuclear Du Pont (Boston, MA). Nitrocellulose ®lters (5 mm pore size) were from Sartorius (Goettingen, FRG.). The dye-reagent concentrate for the determination of protein was obtained from Bio-Rad. All other chemicals used were of reagent grade and were obtained from commercial sources. 2.1. Preparation of synaptosomes and SMV Synaptosomes from adult male Wistar rats were prepared as previously described by Favilli et al. (1994). The vesicles were derived from puri®ed fraction of synaptosomal plasma membranes as reported by Kanner's method (Kanner and Sharon, 1978). The purity of synaptosomes and SMV was determined by electron microscopy observation; no contamination from glial cells was evidenced. The measurement of the speci®c activity of the marker enzymes acetylcholinesterase, Cytocrome c oxidase, NAD+-dependent isocitrate dehydrogenase, and lactate dehydrogenase indicated that the SMV fraction was essentially free of mitochondria and enriched for plasma membranes. In
fact, the activity of acetylcholinesterase, a typical enzyme of the nervous system localized in the external synaptosome membrane, was increased by about 200% compared to that of crude homogenate, while the activity of the other enzymes was reduced by about 100%. Moreover, no signi®cant change in the speci®c activity of acetylcolinesterase was measured when SMV were broken by sonication, indicating that outside-inside vesicles were prevalent. The activities of these enzymes were measured as previously reported (Stio et al., 1993). SMV were loaded with a medium containing 1 mM MgSO4, 1 mM dithiothreitol, 100 mM mannitol, 10 mM Hepes/Tris pH 6.8 and 100 mM KCl (KCl-loaded SMV). In some experiments 100 mM KCl was replaced by 100 mM KH2PO4 or 200 mM mannitol. 2.2. Transport measurements The uptake of radiolabeled substrates was performed by a rapid ®ltration method as described by Vincenzini et al. (1989). Uptake experiments were carried out at 378C by using KCl or KH2PO4 or mannitol-loaded SMV diluted 10-fold with solution containing 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 1 mM dithiothreitol, 100 mM NaCl, 5 mM [3H] GSH, 100 mM GSH in this way a Na+ gradient was imposed. The ®nal volume of transport media was of 100 ml. In some experiments GSH was substituted with 0.4 mM glutamic acid and in others NaCl was replaced isoosmotically by KCl or KH2PO4 or TrisCl or mannitol or NaSCN. The uptake experiments in synaptosomes were performed by suspending the synaptosomes in 0.32 M sucrose and 5 mM Hepes/Tris (pH 7.4) and by diluting three-fold with Krebs-solution containing 1 mM dithiothreitol and 5 mM [3H] GSH, 100 mM GSH or 5 mM [14C]-glutamic acid. In some experiments NaCl of Krebs solution was replaced isoosmotically by KCl and, in others, synaptosomes or SMV were preincubated at 378C for 30 min with 0.5 mM acivicin (AT125), a speci®c inhibitor of gglutamyltranspeptidase (g-GT) (Stole et al. 1994). For trans-stimulation experiments we used KCl-loaded SMV without (control) or with 1 mM unlabeled GSH (GSH-loaded SMV). GSH uptake was carried out at 378C with KCl-loaded SMV and with GSH-loaded SMV diluted 10-fold with solution containing 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl and 5 mM [3H] GSH. In kinetic experiments GSH uptake into KCl-loaded SMV was measured after 5 s of incubation using dierent concentrations of GSH. The time required to stop the uptake was about 1 s. Saturation kinetic and Km and Vmax values were obtained by non-linear regression analysis. Eadie Hofstee plot was also performed. After suitable incubation times, transport of radiolabeled compounds
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2.3. g-GT assay g-GT activity (EC 2.3.2.1) was measured in SMV with a commercially available assay Kit. (Boehringer, Mannheim) using as substrates g-glutamyl-p-nitro-anilide and glycylglycine as previously reported (Favilli, et al. 1994). 2.4. Protein determination Protein concentration was determined by Bradford's method (Bradford, 1976). Bovine serum albumin (Sigma Chemical Company) was used as standard. 2.5. Statistical analyses Statistical analyses were evaluated using Student's ttest by computer program on the Philips PR07BM743. A dierence of P < 0.05 was considered signi®cant. Some data presented were selected from a representative single experiment because of variability between synaptosome preparations. In these cases qualitatively similar results were obtained in separate experiments. 3. Results 3.1. GSH mediated transport in relation to ionic gradients and transmembrane potential
Fig. 1. Time course of GSH and glutamic acid uptake into SMV in the presence of dierent cations. (A) GSH uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 5 mM [3H]GSH, 100 mM unlabeled GSH and 100 mM NaCl (*), or 100 mM KCl (R), or 100 mM TrisCl (Q); (B) Glutamic acid uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 0.4 mM [14C]glutamic acid and 100 mM NaCl (*), or 100 mM KCl (R). The data are the means2SEM of four experiments carried out in triplicate.P < 0.05 in respect to uptake values obtained in presence of NaCl.
was stopped in synaptosomes and SMV by the addition of 3 ml ice-cold Krebs solution or 150 mM NaCl respectively. The mixture was then quickly ®ltered through a nitrocellulose ®lter and washed with 3+3 ml Krebs solution or 150 mM NaCl. Synaptosomes or SMV associated radioactivity found on the ®lter was determined in a liquid scintillation counter. All values were corrected for radioactivity associated with ®lters when uptake mixture was ®ltered without incubation. Other experimental conditions are reported in the legends of Figs. and Tables.
Fig. 1A shows that GSH was taken up into KClloaded SMV in a time-dependent manner and equilibrium was reached very quickly, either in the presence of a Na+ inward gradient (out>in) and a K+ outward gradient (in < out) or of an equal concentration of K+ outside and inside SMV; the behaviour of GSH time courses was similar. Time courses of GSH uptake, when 100 mM GSH was used, showed a steady-state accumulation of approximately 150±190 pmol/mg protein in the presence of Na+ or K+ in extravesicular medium respectively. Considering the previously accepted value of the intravesicular volume (7.4 ml/mg) (Kanner and Sharon, 1978), this accumulation corresponds to a concentration of GSH 20 mM, indicating under these experimental conditions no concentrative uptake within the SMV. However GSH concentration both at short incubation times (15±30 s) and at equilibrium point was about three±four times higher in the presence of extravesicular Na+ or K+ than that measured when these cations were substituted with Tris+. The time course of GSH uptake in the presence of extravesicular Li+ was similar to that observed in the presence of extravesicular Na+ or K+ (data not shown). In order to verify the aspeci®c binding of GSH on SMV, GSH uptake experiments were performed either at 48C or using SMV broken by soni-
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Table 1 Eect of dierent ions on GSH uptake into SMV. GSH uptake was carried out at 378C in KCl or KH2PO4 or mannitol-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 5 mM [3H]GSH, 100 mM unlabeled GSH and 100 mM NaCl or KCl or Tris/HCl or NaH2PO4 or 200 mM mannitol. The data are the means2SEM of three experiments carried out in triplicatea Uptake (pmol GSH/mg protein/5 s) KClin NaClout KClout NaH2PO4out Tris-HClout Mannitolout
7027 9525 80210 3423 4322
KH2PO4in
Mannitolin
6029 9023 7528 3022 4523
150210 110210 13429 4527 4525
a P < 0.05; P < 0.001 in respect to uptake values measured in the presence of NaClout/KClin.
cation. The values of labeled GSH measured at dierent incubation times were similar, and very low when compared to those obtained at 378C; no mediated transport behaviour was observed (data not shown). The functionality of SMV has been demonstrated by studying, the time course of glutamate uptake under the same experimental conditions used for GSH uptake. Fig. 1B shows that glutamate uptake values were higher in the presence of an inward Na+ gradient as compared with those obtained when an equal K+ concentration was present outside and inside SMV. Considering that the membrane enzyme, g-GT, can break down speci®cally g-glutamyl linkage of GSH, we measured the speci®c activity of this enzyme in SMV, but no activity was detected, con®rming that intact GSH fully accounted for vesicles associated radioactivity. In order to con®rm the lack of this enzyme activity on GSH uptake in KCl-loaded SMV we performed a time course of GSH uptake with SMV previously incubated for 30 min with AT125, a speci®c inhibitor of g-GT. No change in GSH uptake values was observed (data not shown). Since various transport systems of aminoacids and their derivatives in the brain are known to be dependent on sodium, potassium or chloride ions, the initial GSH uptake rate into SMV was tested after 15 s of incubation in the presence of dierent ions in the intra/extra vesicular medium. It is evident in Table 1 that the replacement of KCl with KH2PO4 in the intravesicular medium did not signi®cantly change the GSH uptake values in presence of all extravesicular media utilized. The lowest values were measured when Tris+ or mannitol were present in extravesicular medium as compared to GSH uptake values measured in the presence of extravesicular K+ or Na+. The replacement of KCl with mannitol in the intravesicular medium similarly decreased GSH uptake when Tris+ or mannitol were in the
Fig. 2. Eect of ionophores on GSH uptake into SMV. GSH uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 5 mM [3H]GSH, 100 mM unlabeled GSH 100 mM, NaCl and 0.5% ethanol without (*), or with 5 mM nigericin (R) or 4 mM valinomycin (Q). The data are the means2SEM of four experiments carried out in triplicate.P < 0.05; P < 0.001 in respect to uptake values obtained in presence of NaCl without ionophores.
extravesicular medium, while GSH uptake remarkably increased in the presence either of Na+ or K+ gradient (out>in). GSH is negatively charged at physiological pH, therefore its transport may be aected both by potential dierences across synaptosomal membranes and by the presence of ions. To con®rm this hypothesis, the time-course of GSH uptake into KCl-loaded SMV was studied in the presence of an interior negative membrane potential induced by valynomicin and an outward K+ gradient; Fig. 2 shows that this potential change signi®cantly decreased GSH uptake at various incubation times. Similar decreases of GSH uptake values were also obtained when extravesicular Clÿ was substituted with the more permeant anion SCNÿ, which hyperpolarizes the electrical membrane potential (data not shown). Moreover, Fig. 2 shows a remarkable decrease of GSH uptake values in the presence of nigericin, an ionophore that dissipates the electrochemical sodium gradient, Similar results were obtained studying the time-course of GSH uptake into intact synaptosomes (data not shown). The subsequent experiments to characterize GSH mediated transport have been performed using an inward Na+ gradient and an outward K+ gradient, considering that these experimental conditions are similar to those of the physiological synaptosome environment, and that they have been used to study other metabolite transport systems in synaptosomes.
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Fig. 3. Eect of varying GSH concentrations on the initial rate of GSH uptake into SMV. GSH uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl and varying concentrations of [3H]GSH. The saturation kinetic is obtained by non linear regression, i.e. by ®tting the equation v=Vmax [S]/(Km+[S]) to the experimental data. The data are the means2SEM of three experiments performed in triplicate. Km=4.520.8 mM; Vmax=7028.5 pmol/5 s per mg protein.
3.2. Kinetic data of GSH uptake In order to con®rm whether the vesicle associated radioactivity really re¯ected a carrier mediated transport the initial rate of GSH uptake was determined at dierent GSH concentrations. Since the linearity with incubation time was observed up to 15 s, GSH uptake was measured after 5 s of incubation. The obtained behaviour of GSH uptake was hyperbolic and was consistent with a Michaelis-Menten saturation kinetic, (with Km of 4.520.8 mM and Vmax of 7028.5 pmol GSH/mg protein/ 5 s) Fig. 3. 3.3. Trans-stimulation of GSH uptake A typical trans-stimulation experiment with unlabeled GSH to demonstrate the evidence for carrierTable 2 Trans-stimulation of GSH uptake into SMV. GSH uptake was carried out at 378C in KCl-loaded SMV without (control) or with 1 mM unlabeled GSH (GSH-loaded SMV) were diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl and 5 mM [3H]GSH. The data are the means2SEM of three experiments carried out in triplicatea Uptake (pmol GSH/mg protein) Incubation (Time)
control
GSH-loaded SMV
15 s 5 min
7027 150220
a
P < 0.05 in respect to control values.
160220 170225
513
Fig. 4. Eect of medium osmolarity on GSH uptake into SMV. GSH uptake values were determined after 20 min of incubation at 378C in KCl-loaded SMV diluted 10-fold with 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl, 5 mM [3H]GSH, 100 mM unlabeled GSH and varying concentrations of mannitol (100±800 mM). The osmolarity ratios inside/outside the vesicles are reported on the abscissa. The y intercept shows that binding of GSH in the vesicles is 28%.
mediated transport in SMV was performed. Table 2 shows signi®cant increase in the initial rate of labeled GSH uptake, of about 2.3 times after 15 s of incubation, in GSH-loaded SMV as compared with KClloaded SMV without unlabeled GSH (control). The increase of GSH uptake value was not due to volume variation, considering that the equilibrium values measured after 5 min of incubation were similar. 3.4. GSH binding In order to distinguish between the GSH binding to the vesicle membrane surface and GSH transport into KCl-loaded SMV, the eect of medium osmolarity on equilibrium GSH uptake values (after 20 min of incubation) was observed. The data shown in Fig. 4 indicate that when the ratio of osmolarity inside/ osmolarity outside the vesicles decreased (with consequent decrease of vesicle volume), GSH intravesicular content also decreased. The GSH uptake value obtained from extrapolation to a zero intercept (in®nite osmolarity outside the vesicle) shows that GSH binding is 28% of total vesicle-associated GSH.
4. Discussion The presence of GSH mediated transport in synaptosome membranes of rat cerebral cortex has been demonstrated for the ®rst time in this study. Dierent lines of evidence suggest that GSH uptake represents
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mediated transport and not a binding artefact: (1) fast uptake at short incubation time, as observed in the time-course of some neurotransmitter or aminoacid uptake in SMV (Kanner and Sharon, 1978; Tan and Ng, 1995); (2) the Michaelis-Menten saturation kinetics that indicates the presence of a single GSH mediated transport; (3) the decrease of GSH accumulation in SMV when the osmolarity of the medium is increased; (4) the trans-stimulation eect on GSH uptake in GSH-loaded SMV; an analogous observation was also utilized to con®rm the presence of GSH-mediated transport in vesicles obtained by using membranes of dierent types of cells (GarcõÂ a-Ruiz et al., 1992; Vincenzini et al., 1991). The ®ndings obtained indicate that GSH transport in SMV is activated by the presence of either an inward gradient of cations, like Na+ and K+, or of a membrane depolarization (AragoÂn et al., 1988). An involvement of Na+ gradient in GSH uptake is also demonstrated by nigericin experiments. The eect of Na+ and K+ in GSH uptake is not due to the presence of their positive charge alone, but rather to their ability to cross the membrane, unlike Tris+, cation to which the membranes are not permeable. Experiments performed by valinomycininduced K+ diusion potential con®rm the in¯uence of membrane potential changes on GSH uptake, similar to that found in other GSH transport systems characterized in dierent tissues (Vincenzini et al., 1989) and they are consistent with a Na+-independent electrogenic GSH transport by a negative charge transfer. We should also note that in the presence of an inward Na+ gradient GSH uptake values double when outward K+ gradient is absent and inside K+ is replaced with mannitol. Considering the known high permeability of synaptosomal membrane to K+ (AragoÂn et al., 1988), an outward K+ gradient can create a K+ eux and an interior negative potential which reduces GSH uptake. It is likely that an inward cation gradient activates GSH transport by causing transmembrane potential which is optimal for activity of GSH transporter, as recently reported for the transport of g-aminobutyric acid by synaptic plasma membrane vesicles isolated from sheep brain cortex (Rahman et al., 1996). A direct interaction of extravesicular cations with GSH and/or with transporter is also possible. In order to explain the electrogenic transport of GSH we can hypothesise that this may occur simultaneously with the uptake of both Na+ and Clÿ or with Na+-K+ antiport. This or other possibilities must be investigated further: direct ¯ux measurements are required to clarify the exact role of ions in GSH transport in SMV. In conclusion, the data obtained exclude a typical Na+-dependent transport of GSH similar to that observed for glutamate, and they indicate that both cations gradients and po-
tential changes can activate GSH transport system and constitute in vivo an energetic force for GSH uptake. Considering that intracellular concentration of GSH measured in the brain is higher than extracellular concentration (Juurlink et al., 1996) GSH uptake may occur only by activated transport. However, it is dicult to relate the eective functionality of synaptosomal GSH transporter to the intra and extra concentration of GSH in synaptosomes, specialized structures, in which the concentrations of various metabolites may change remarkably and GSH content also in physiological conditions is not known. The ability of internal GSH to trans-stimulate its uptake through the membrane con®rms the presence of mediated and bidirectional transport system. This activation eect due to the acceleration of external substrate transport into cells by internal substrate is interpreted as a counter-exchange of the external with the internal substrate by transporter (Kessler and Toggenburger, 1979). Therefore we can also hypotize an antiport with other substrates and the possibility of either uptake or eux of GSH in relation to physiological conditions. It is also evident that changes in ionic ¯uxes and in the membrane potential involved in the polarization and depolarization of nerve endings may modulate and regulate the activity of GSH transporter. These characteristics and the low Km value of GSH synaptosomal transporter are similar to those of the transport systems of neurotransmitter aminoacids in synaptosome, which operate in eux direction under depolarization conditions, and in in¯ux direction during repolarization (AragoÂn et al., 1988; Kanner and Sharon, 1978; Tan and Ng, 1995; Dowd et al., 1996). Eectively, other authors report that in rat brain slices GSH is released upon K+ depolarization (Ogita et al., 1995; ZaÈngerle et al., 1992). They suggest that GSH originates from neuronal compartment, since this release is Ca2+-dependent, and that it may modulate neuronal excitation through interaction with receptors for excitatory aminoacids. The percentage of GSH binding measured at the equilibrium point may con®rm the presence of binding sites for GSH in synaptosomal membrane receptors, as also reported by others (Ogita and Yoneda, 1989; Guo et al., 1992). These data may indicate an involvement of this transporter in the role suggested for GSH in synaptic neurotransmission, (Guo et al., 1992). An other possible physiological role of GSH synaptosomal transporter may be to supply neurons with GSH. Moreover, this transporter, together with those found by Kannan et al. (1996) in bovine brain capillary and by Sagara et al. (1996) in astroglial cells, may contribute to maintaining a GSH interorgan cycle as well as GSH homeostasis. Recently it has been suggested that neuronal GSH metabolism is linked to that in glial cells. GSH levels in astrocytes are higher than those measured in neurons (Sagara et
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al., 1996; Dringen et al., 1997; Juurlink et al., 1996) and it has been demonstrated that GSH is released from astrocites by mediated transport, and that stress oxidative increases GSH eux (Sagara et al., 1996). Increases of GSH extracellular levels in the brain have also been measured by microdyalisis experiments in ischemic rat striatum (Orwar et al., 1994). Therefore it has been suggested that the release of GSH from astroglial cells may contribute to protect neurons against oxidative injury and to the maintenance of the GSH neuronal pool by the gGT breakdown of extracellular GSH, subsequent uptake of its constituent aminoacids and intracellular GSH resynthesis (Sagara et al., 1996; Dringen et al., 1997). These authors exclude a direct GSH uptake into neurons, but no experimental data are reported. Moreover, we do not detect g-GT activity in synaptosomes. The kinetic parameter values of GSH eux from astrocytes (Km 36 mM and Vmax 0.39 nmol/ min/mg protein) are very dierent from those measured for GSH uptake in SMV, indicating the presence of two dierent types of GSH transporters, which probably correspond to dierent physiological roles. The high uptake rate and anity for GSH of synaptosomal transporter suggest that astrocytes may eectively supply neurons with GSH. This would be important, considering that GSH is involved in regulating levels of neuroactive aminoacids and that low levels of GSH and of g-glutamylcysteine synthetase expression have been found in cerebral cortex (Kang et al., 1997). Changes in GSH synthesis and a decrease in GSH content in cerebral cortex have been related to susceptibility to neuropathologies often associated with oxidative stress (Cassarino et al., 1997). Acknowledgements This work was supported by the Italian Association for Cancer Research (AIRC) and by the Ministero della Pubblica Istruzione e della Ricerca Scienti®ca e Tecnologica (40%). References AragoÂn, M.C., AgulloÂ, L., GimeÂnez, C., 1988. Depolarizationinduced release of glycine and D-alanine from plasma membrane vesicles derived from rat brain synaptosomes. Biochimica Biophysica Acta 941, 209±216. Bains, J.S., Shaw, C.A., 1997. Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Research Reviews 25, 335±358. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248±254. Cassarino, D.S., Fall, C.P., Swerdlow, R.H., Smith, T.S., Halvorsen, E.M., Miller, S.W., Parks, J.P., Parker Jr, W.D., Bennett Jr, J.P.,
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1995. A possible role of glutathione as an endogenous agonist at the N-methyl-D-aspartate recognition domain in rat brain. Journal of Neurochemistry 64, 1088±1096. Orlowsky, M., Karkowsky, A., 1976. Glutathione metabolism and some possible functions of glutathione in the nervous system. International Review of Neurobiology 19, 75±81. Orwar, O., Li, X., AndineÂ, P., BergstroÈm, C.M., Hagberg, H., Folestad, S., Sandberg, M., 1994. Increased intra- and extracellular concentrations of g-glutamylglutamate and related dipeptides in the ischemic rat striatum: involvement of g-glutamyl transpeptidase. Journal of Neurochemistry 63, 1371±1376. Perry, T.L., Yong, V.W., 1987. Amino acids, glutathione, and glutathione transferase activity in the brains of patients with Alzheimer's disease. Annals Neurology 21, 331±336. Pileblad, E., Magnusson, T., Fornstedt, B., 1989. Reduction of brain glutathione by L-buthionine sulfoximine potentiates the dopamine-depleting action of 6-hydroxydopamine in rat striatum. Journal of Neurochemistry 52, 978±980. Pileblad, E., Eriksson, P.S., Hansson, E., 1991. The presence of glutathione in primary neuronal and astroglial cultures from rat cerebral cortex and brain stem. Journal of Neural Transmission 86, 43±49. Rahman, I., Bel, A., Mulier, B., Lawson, M.F., Harrison, D.J., MacNee, W., Smith, C.A.D., 1996. Transcriptional regulation of g-glutamylcysteine synthetase-heavy subunit by oxidants in human alveolar epithelial cells. Biochemical Biophysical Research Communication 229, 82±837. Raps, S.P., Lai, J.C.K., Hertz, L., Cooper, A.J.L., 1989. Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons. Brain Research 493, 398±401.
Sagara, J., Makino, N., Bannai, S., 1996. Glutathione eux cultured astrocytes. Journal of Neurochemistry 66, 1876±1881. Stio, M., Lunghi, B., Iantomasi, T., Vincenzini, M.T., Treves, C., 1993. Eect of vitamin D de®ciency and 1,25-dihydroxyvitamin D3 on metabolism and D-glucose transport in rat cerebral cortex. Journal of Neuroscience Research 35, 559±566. Stole, E., Smith, T.K., Manning, J.M., Meister, A., 1994. Interaction of g-glutamyl transpeptidase with acivicin. Journal of Biological Chemistry 269, 21435±21439. Tan, C.H., Ng, F.H., 1995. High and low anity transport of L-arginine in rat brain synaptosomes. Experientia 51, 1052±1054. Vincenzini, M.T., Iantomasi, T., Favilli, F., 1989. Glutathione transport across intestinal brush-border membranes: eect of ions, pH, DC, and inhibitors. Biochimica Biophysica Acta 987, 29±37. Vincenzini, M.T., Favilli, F., Stio, M., Iantomasi, T., 1991. Intestinal glutathione transport system: a possible detoxication role. Biochimica Biophysica Acta 1073, 571±579. Yudko, M., Pleasure, D., Cregar, L., Lin, Z., Nissim, I., Stern, J., Nissim, I., 1990. Glutathione turnover in cultured astrocytes: studies with [15N]glutamate. Journal of Neurochemistry 55, 137± 145. ZaÈngerle, L., CueÂnod, M., Winterhalter, K.H., Quang, Do K., 1992. Screening of thiol compounds: depolarization-induced release of glutathione and cysteine from rat brain slices. Journal Neurochemistry 59, 181±189. Zlokovic, B.V., Mackic, J.B., McComb, J.G., Weiss, M.H., Kaplowitz, N., 1994. Evidence for transcapillary transport of reduced glutathione in vascular perfused guinea-pig brain. Biochemical Biophysical Research Comunication 201, 402±408.
Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles Teresa Iantomasi, Fabio Favilli, Maria T. Vincenzini* Department of Biochemical Sciences, University of Firenze, viale Morgagni 50, Firenze, 50134 Italy Received 1 October 1998; received in revised form 1 February 1999; accepted 1 February 1999
Abstract Glutathione (GSH) transport was studied in synaptosomal membrane vesicles (SMV) of rat cerebral cortex. The present study shows that GSH uptake into SMV occurs very quickly in a time-dependent manner into an osmotically active intravesicular space. The initial rate of transport followed Michealis-Menten saturation kinetics with a Km 4.5 2 0.8 mM that shows a high anity of the transporter for GSH. Therefore GSH uptake in SMV occurs by a mediated transport system which can be activated by either an inward gradient of cations, like Na+ or K+, or membrane depolarization. These results, together with those obtained by valinomycin-induced K+ diusion potential, indicate that GSH synaptosomal transport is electrogenic by a negative charge transfer. The increase of GSH uptake measured by trans-stimulation experiments con®rms a GSH bidirectional mediated transport which seems susceptible of modulation by changes in ionic ¯uxes and in the membrane potential. These results may indicate a possible involvement of this transporter in the role suggested for GSH in synaptic neurotransmission; also considering that GSH precursor of neuroactive aminoacids (glyeine, glutamate), may contribute to regulate their level in synapses. Finally, a GSH transporter in synaptosomes may contribute to maintaining the GSH homeostasis in cerebral cortex, where decreases of GSH levels have been related to susceptibility to neuropathologies. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Glutathione transporter; Rat; Synaptosomes membranes
1. Introduction GSH is a major and ubiquitous intracellular antioxidant (Ishikawa and Sies, 1989). GSH concentrations in the brain of dierent animals are high, ranging from 0.5 to 3 mM (Orlowsky and Karkowsky, 1976; Sagara et al., 1996). GSH has been found prevalently in primary astrocyte culture (Huang and Philbert, 1995; Sagara et al., 1996; Juurlink et al., 1996) while little GSH is present in neurones (Raps et al., 1989) and some studies have also demonstrated a compartmentaAbbreviations: SMV, synaptosomal membrane vesicles; g-GT, gglutamyltranspeptidase; AT125, Acivicin (LaS; 5 S)-a-amino-3chloro4,5-dihydro-5-isoxozoleacetic acid). * Corresponding author. Tel.: +39-55-416686; fax: +39-554222725. E-mail address: [email protected]®.it (M.T. Vincenzini)
lisation of GSH and GSH-related enzymes within neurones with GSH accumulation in axon terminals in synaptosomal fraction (Pileblad et al., 1991; Favilli et al., 1994). GSH antioxidant and detoxi®cating action in brain tissue and particularly in synaptosomes has been established (Pileblad et al., 1989; Cooper and Kristal, 1997), together with pathological changes of GSH metabolism associated with neurodegenerative processes of the brain (Perry and Yong, 1987; Mizui et al., 1992; Bains and Shaw, 1997). However, the speci®c physiological role of GSH in the brain still remains to be explained. Several recent studies have suggested a possible additional role for GSH as a neuromodulator and/or neurotransmitter (Guo et al., 1992; Lanius et al., 1994; Ogita et al., 1995). In fact, there is evidence that GSH may in¯uence neurotransmission by interacting directly with the speci®c binding site of glutamic acid on the synaptic membranes and/or as precursor
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of neurotransmitter aminoacids (Ogita et al., 1995). GSH-speci®c binding sites in brain synaptic membranes and in cultured astrocytes have been found (Ogita and Yoneda, 1989; Guo et al., 1992). Moreover, it has been demonstrated that both GSH and cystine are released into perfusates from rat brain slices on stimulation by K+ in a Ca2+ dependent manner (ZaÈngerle et al., 1992), and astrocytes are also known to eux GSH at a high rate (Yudko et al., 1990; Sagara et al., 1996; Dringen et al., 1997). Previous data have also demonstrated GSH transport in rat blood-brain barrier and in perfused guinea pig brain (Kannan et al., 1990; Zlokovic et al., 1994), and recently three distinct GSH transporter activities have been identi®ed in bovine brain capillary (Kannan et al., 1996). Many data indicate that synaptosomes contain a heterogeneous population of transporters of aminoacids and their precursors and that uptake processes are in some cases the primary mechanism for the neurotransmitters remotion from the synapse (Dowd et al., 1996). In view of these previous ®ndings, we investigated whether GSH can cross SMV of rat cerebral cortex by mediated transport in order to modulate cerebral GSH turnover, levels of neuroactive aminoacids (L-glutamate, glycine and L-cysteine) and to regulate mechanisms of neurotransmission. 2. Experimental procedures [Glycine-2,3H] GSH (spec. act. 43.8 Ci/mmol) and acid glutamic, L-[14C(U)] (spec. act. 281.4 mCi/mmol) were obtained from New England Nuclear Du Pont (Boston, MA). Nitrocellulose ®lters (5 mm pore size) were from Sartorius (Goettingen, FRG.). The dye-reagent concentrate for the determination of protein was obtained from Bio-Rad. All other chemicals used were of reagent grade and were obtained from commercial sources. 2.1. Preparation of synaptosomes and SMV Synaptosomes from adult male Wistar rats were prepared as previously described by Favilli et al. (1994). The vesicles were derived from puri®ed fraction of synaptosomal plasma membranes as reported by Kanner's method (Kanner and Sharon, 1978). The purity of synaptosomes and SMV was determined by electron microscopy observation; no contamination from glial cells was evidenced. The measurement of the speci®c activity of the marker enzymes acetylcholinesterase, Cytocrome c oxidase, NAD+-dependent isocitrate dehydrogenase, and lactate dehydrogenase indicated that the SMV fraction was essentially free of mitochondria and enriched for plasma membranes. In
fact, the activity of acetylcholinesterase, a typical enzyme of the nervous system localized in the external synaptosome membrane, was increased by about 200% compared to that of crude homogenate, while the activity of the other enzymes was reduced by about 100%. Moreover, no signi®cant change in the speci®c activity of acetylcolinesterase was measured when SMV were broken by sonication, indicating that outside-inside vesicles were prevalent. The activities of these enzymes were measured as previously reported (Stio et al., 1993). SMV were loaded with a medium containing 1 mM MgSO4, 1 mM dithiothreitol, 100 mM mannitol, 10 mM Hepes/Tris pH 6.8 and 100 mM KCl (KCl-loaded SMV). In some experiments 100 mM KCl was replaced by 100 mM KH2PO4 or 200 mM mannitol. 2.2. Transport measurements The uptake of radiolabeled substrates was performed by a rapid ®ltration method as described by Vincenzini et al. (1989). Uptake experiments were carried out at 378C by using KCl or KH2PO4 or mannitol-loaded SMV diluted 10-fold with solution containing 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 1 mM dithiothreitol, 100 mM NaCl, 5 mM [3H] GSH, 100 mM GSH in this way a Na+ gradient was imposed. The ®nal volume of transport media was of 100 ml. In some experiments GSH was substituted with 0.4 mM glutamic acid and in others NaCl was replaced isoosmotically by KCl or KH2PO4 or TrisCl or mannitol or NaSCN. The uptake experiments in synaptosomes were performed by suspending the synaptosomes in 0.32 M sucrose and 5 mM Hepes/Tris (pH 7.4) and by diluting three-fold with Krebs-solution containing 1 mM dithiothreitol and 5 mM [3H] GSH, 100 mM GSH or 5 mM [14C]-glutamic acid. In some experiments NaCl of Krebs solution was replaced isoosmotically by KCl and, in others, synaptosomes or SMV were preincubated at 378C for 30 min with 0.5 mM acivicin (AT125), a speci®c inhibitor of gglutamyltranspeptidase (g-GT) (Stole et al. 1994). For trans-stimulation experiments we used KCl-loaded SMV without (control) or with 1 mM unlabeled GSH (GSH-loaded SMV). GSH uptake was carried out at 378C with KCl-loaded SMV and with GSH-loaded SMV diluted 10-fold with solution containing 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl and 5 mM [3H] GSH. In kinetic experiments GSH uptake into KCl-loaded SMV was measured after 5 s of incubation using dierent concentrations of GSH. The time required to stop the uptake was about 1 s. Saturation kinetic and Km and Vmax values were obtained by non-linear regression analysis. Eadie Hofstee plot was also performed. After suitable incubation times, transport of radiolabeled compounds
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2.3. g-GT assay g-GT activity (EC 2.3.2.1) was measured in SMV with a commercially available assay Kit. (Boehringer, Mannheim) using as substrates g-glutamyl-p-nitro-anilide and glycylglycine as previously reported (Favilli, et al. 1994). 2.4. Protein determination Protein concentration was determined by Bradford's method (Bradford, 1976). Bovine serum albumin (Sigma Chemical Company) was used as standard. 2.5. Statistical analyses Statistical analyses were evaluated using Student's ttest by computer program on the Philips PR07BM743. A dierence of P < 0.05 was considered signi®cant. Some data presented were selected from a representative single experiment because of variability between synaptosome preparations. In these cases qualitatively similar results were obtained in separate experiments. 3. Results 3.1. GSH mediated transport in relation to ionic gradients and transmembrane potential
Fig. 1. Time course of GSH and glutamic acid uptake into SMV in the presence of dierent cations. (A) GSH uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 5 mM [3H]GSH, 100 mM unlabeled GSH and 100 mM NaCl (*), or 100 mM KCl (R), or 100 mM TrisCl (Q); (B) Glutamic acid uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 0.4 mM [14C]glutamic acid and 100 mM NaCl (*), or 100 mM KCl (R). The data are the means2SEM of four experiments carried out in triplicate.P < 0.05 in respect to uptake values obtained in presence of NaCl.
was stopped in synaptosomes and SMV by the addition of 3 ml ice-cold Krebs solution or 150 mM NaCl respectively. The mixture was then quickly ®ltered through a nitrocellulose ®lter and washed with 3+3 ml Krebs solution or 150 mM NaCl. Synaptosomes or SMV associated radioactivity found on the ®lter was determined in a liquid scintillation counter. All values were corrected for radioactivity associated with ®lters when uptake mixture was ®ltered without incubation. Other experimental conditions are reported in the legends of Figs. and Tables.
Fig. 1A shows that GSH was taken up into KClloaded SMV in a time-dependent manner and equilibrium was reached very quickly, either in the presence of a Na+ inward gradient (out>in) and a K+ outward gradient (in < out) or of an equal concentration of K+ outside and inside SMV; the behaviour of GSH time courses was similar. Time courses of GSH uptake, when 100 mM GSH was used, showed a steady-state accumulation of approximately 150±190 pmol/mg protein in the presence of Na+ or K+ in extravesicular medium respectively. Considering the previously accepted value of the intravesicular volume (7.4 ml/mg) (Kanner and Sharon, 1978), this accumulation corresponds to a concentration of GSH 20 mM, indicating under these experimental conditions no concentrative uptake within the SMV. However GSH concentration both at short incubation times (15±30 s) and at equilibrium point was about three±four times higher in the presence of extravesicular Na+ or K+ than that measured when these cations were substituted with Tris+. The time course of GSH uptake in the presence of extravesicular Li+ was similar to that observed in the presence of extravesicular Na+ or K+ (data not shown). In order to verify the aspeci®c binding of GSH on SMV, GSH uptake experiments were performed either at 48C or using SMV broken by soni-
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Table 1 Eect of dierent ions on GSH uptake into SMV. GSH uptake was carried out at 378C in KCl or KH2PO4 or mannitol-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 5 mM [3H]GSH, 100 mM unlabeled GSH and 100 mM NaCl or KCl or Tris/HCl or NaH2PO4 or 200 mM mannitol. The data are the means2SEM of three experiments carried out in triplicatea Uptake (pmol GSH/mg protein/5 s) KClin NaClout KClout NaH2PO4out Tris-HClout Mannitolout
7027 9525 80210 3423 4322
KH2PO4in
Mannitolin
6029 9023 7528 3022 4523
150210 110210 13429 4527 4525
a P < 0.05; P < 0.001 in respect to uptake values measured in the presence of NaClout/KClin.
cation. The values of labeled GSH measured at dierent incubation times were similar, and very low when compared to those obtained at 378C; no mediated transport behaviour was observed (data not shown). The functionality of SMV has been demonstrated by studying, the time course of glutamate uptake under the same experimental conditions used for GSH uptake. Fig. 1B shows that glutamate uptake values were higher in the presence of an inward Na+ gradient as compared with those obtained when an equal K+ concentration was present outside and inside SMV. Considering that the membrane enzyme, g-GT, can break down speci®cally g-glutamyl linkage of GSH, we measured the speci®c activity of this enzyme in SMV, but no activity was detected, con®rming that intact GSH fully accounted for vesicles associated radioactivity. In order to con®rm the lack of this enzyme activity on GSH uptake in KCl-loaded SMV we performed a time course of GSH uptake with SMV previously incubated for 30 min with AT125, a speci®c inhibitor of g-GT. No change in GSH uptake values was observed (data not shown). Since various transport systems of aminoacids and their derivatives in the brain are known to be dependent on sodium, potassium or chloride ions, the initial GSH uptake rate into SMV was tested after 15 s of incubation in the presence of dierent ions in the intra/extra vesicular medium. It is evident in Table 1 that the replacement of KCl with KH2PO4 in the intravesicular medium did not signi®cantly change the GSH uptake values in presence of all extravesicular media utilized. The lowest values were measured when Tris+ or mannitol were present in extravesicular medium as compared to GSH uptake values measured in the presence of extravesicular K+ or Na+. The replacement of KCl with mannitol in the intravesicular medium similarly decreased GSH uptake when Tris+ or mannitol were in the
Fig. 2. Eect of ionophores on GSH uptake into SMV. GSH uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 5 mM [3H]GSH, 100 mM unlabeled GSH 100 mM, NaCl and 0.5% ethanol without (*), or with 5 mM nigericin (R) or 4 mM valinomycin (Q). The data are the means2SEM of four experiments carried out in triplicate.P < 0.05; P < 0.001 in respect to uptake values obtained in presence of NaCl without ionophores.
extravesicular medium, while GSH uptake remarkably increased in the presence either of Na+ or K+ gradient (out>in). GSH is negatively charged at physiological pH, therefore its transport may be aected both by potential dierences across synaptosomal membranes and by the presence of ions. To con®rm this hypothesis, the time-course of GSH uptake into KCl-loaded SMV was studied in the presence of an interior negative membrane potential induced by valynomicin and an outward K+ gradient; Fig. 2 shows that this potential change signi®cantly decreased GSH uptake at various incubation times. Similar decreases of GSH uptake values were also obtained when extravesicular Clÿ was substituted with the more permeant anion SCNÿ, which hyperpolarizes the electrical membrane potential (data not shown). Moreover, Fig. 2 shows a remarkable decrease of GSH uptake values in the presence of nigericin, an ionophore that dissipates the electrochemical sodium gradient, Similar results were obtained studying the time-course of GSH uptake into intact synaptosomes (data not shown). The subsequent experiments to characterize GSH mediated transport have been performed using an inward Na+ gradient and an outward K+ gradient, considering that these experimental conditions are similar to those of the physiological synaptosome environment, and that they have been used to study other metabolite transport systems in synaptosomes.
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Fig. 3. Eect of varying GSH concentrations on the initial rate of GSH uptake into SMV. GSH uptake was carried out at 378C in KCl-loaded SMV diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl and varying concentrations of [3H]GSH. The saturation kinetic is obtained by non linear regression, i.e. by ®tting the equation v=Vmax [S]/(Km+[S]) to the experimental data. The data are the means2SEM of three experiments performed in triplicate. Km=4.520.8 mM; Vmax=7028.5 pmol/5 s per mg protein.
3.2. Kinetic data of GSH uptake In order to con®rm whether the vesicle associated radioactivity really re¯ected a carrier mediated transport the initial rate of GSH uptake was determined at dierent GSH concentrations. Since the linearity with incubation time was observed up to 15 s, GSH uptake was measured after 5 s of incubation. The obtained behaviour of GSH uptake was hyperbolic and was consistent with a Michaelis-Menten saturation kinetic, (with Km of 4.520.8 mM and Vmax of 7028.5 pmol GSH/mg protein/ 5 s) Fig. 3. 3.3. Trans-stimulation of GSH uptake A typical trans-stimulation experiment with unlabeled GSH to demonstrate the evidence for carrierTable 2 Trans-stimulation of GSH uptake into SMV. GSH uptake was carried out at 378C in KCl-loaded SMV without (control) or with 1 mM unlabeled GSH (GSH-loaded SMV) were diluted 10-fold with 100 mM mannitol, 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl and 5 mM [3H]GSH. The data are the means2SEM of three experiments carried out in triplicatea Uptake (pmol GSH/mg protein) Incubation (Time)
control
GSH-loaded SMV
15 s 5 min
7027 150220
a
P < 0.05 in respect to control values.
160220 170225
513
Fig. 4. Eect of medium osmolarity on GSH uptake into SMV. GSH uptake values were determined after 20 min of incubation at 378C in KCl-loaded SMV diluted 10-fold with 10 mM Hepes/Tris (pH 6.8), 100 mM NaCl, 5 mM [3H]GSH, 100 mM unlabeled GSH and varying concentrations of mannitol (100±800 mM). The osmolarity ratios inside/outside the vesicles are reported on the abscissa. The y intercept shows that binding of GSH in the vesicles is 28%.
mediated transport in SMV was performed. Table 2 shows signi®cant increase in the initial rate of labeled GSH uptake, of about 2.3 times after 15 s of incubation, in GSH-loaded SMV as compared with KClloaded SMV without unlabeled GSH (control). The increase of GSH uptake value was not due to volume variation, considering that the equilibrium values measured after 5 min of incubation were similar. 3.4. GSH binding In order to distinguish between the GSH binding to the vesicle membrane surface and GSH transport into KCl-loaded SMV, the eect of medium osmolarity on equilibrium GSH uptake values (after 20 min of incubation) was observed. The data shown in Fig. 4 indicate that when the ratio of osmolarity inside/ osmolarity outside the vesicles decreased (with consequent decrease of vesicle volume), GSH intravesicular content also decreased. The GSH uptake value obtained from extrapolation to a zero intercept (in®nite osmolarity outside the vesicle) shows that GSH binding is 28% of total vesicle-associated GSH.
4. Discussion The presence of GSH mediated transport in synaptosome membranes of rat cerebral cortex has been demonstrated for the ®rst time in this study. Dierent lines of evidence suggest that GSH uptake represents
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mediated transport and not a binding artefact: (1) fast uptake at short incubation time, as observed in the time-course of some neurotransmitter or aminoacid uptake in SMV (Kanner and Sharon, 1978; Tan and Ng, 1995); (2) the Michaelis-Menten saturation kinetics that indicates the presence of a single GSH mediated transport; (3) the decrease of GSH accumulation in SMV when the osmolarity of the medium is increased; (4) the trans-stimulation eect on GSH uptake in GSH-loaded SMV; an analogous observation was also utilized to con®rm the presence of GSH-mediated transport in vesicles obtained by using membranes of dierent types of cells (GarcõÂ a-Ruiz et al., 1992; Vincenzini et al., 1991). The ®ndings obtained indicate that GSH transport in SMV is activated by the presence of either an inward gradient of cations, like Na+ and K+, or of a membrane depolarization (AragoÂn et al., 1988). An involvement of Na+ gradient in GSH uptake is also demonstrated by nigericin experiments. The eect of Na+ and K+ in GSH uptake is not due to the presence of their positive charge alone, but rather to their ability to cross the membrane, unlike Tris+, cation to which the membranes are not permeable. Experiments performed by valinomycininduced K+ diusion potential con®rm the in¯uence of membrane potential changes on GSH uptake, similar to that found in other GSH transport systems characterized in dierent tissues (Vincenzini et al., 1989) and they are consistent with a Na+-independent electrogenic GSH transport by a negative charge transfer. We should also note that in the presence of an inward Na+ gradient GSH uptake values double when outward K+ gradient is absent and inside K+ is replaced with mannitol. Considering the known high permeability of synaptosomal membrane to K+ (AragoÂn et al., 1988), an outward K+ gradient can create a K+ eux and an interior negative potential which reduces GSH uptake. It is likely that an inward cation gradient activates GSH transport by causing transmembrane potential which is optimal for activity of GSH transporter, as recently reported for the transport of g-aminobutyric acid by synaptic plasma membrane vesicles isolated from sheep brain cortex (Rahman et al., 1996). A direct interaction of extravesicular cations with GSH and/or with transporter is also possible. In order to explain the electrogenic transport of GSH we can hypothesise that this may occur simultaneously with the uptake of both Na+ and Clÿ or with Na+-K+ antiport. This or other possibilities must be investigated further: direct ¯ux measurements are required to clarify the exact role of ions in GSH transport in SMV. In conclusion, the data obtained exclude a typical Na+-dependent transport of GSH similar to that observed for glutamate, and they indicate that both cations gradients and po-
tential changes can activate GSH transport system and constitute in vivo an energetic force for GSH uptake. Considering that intracellular concentration of GSH measured in the brain is higher than extracellular concentration (Juurlink et al., 1996) GSH uptake may occur only by activated transport. However, it is dicult to relate the eective functionality of synaptosomal GSH transporter to the intra and extra concentration of GSH in synaptosomes, specialized structures, in which the concentrations of various metabolites may change remarkably and GSH content also in physiological conditions is not known. The ability of internal GSH to trans-stimulate its uptake through the membrane con®rms the presence of mediated and bidirectional transport system. This activation eect due to the acceleration of external substrate transport into cells by internal substrate is interpreted as a counter-exchange of the external with the internal substrate by transporter (Kessler and Toggenburger, 1979). Therefore we can also hypotize an antiport with other substrates and the possibility of either uptake or eux of GSH in relation to physiological conditions. It is also evident that changes in ionic ¯uxes and in the membrane potential involved in the polarization and depolarization of nerve endings may modulate and regulate the activity of GSH transporter. These characteristics and the low Km value of GSH synaptosomal transporter are similar to those of the transport systems of neurotransmitter aminoacids in synaptosome, which operate in eux direction under depolarization conditions, and in in¯ux direction during repolarization (AragoÂn et al., 1988; Kanner and Sharon, 1978; Tan and Ng, 1995; Dowd et al., 1996). Eectively, other authors report that in rat brain slices GSH is released upon K+ depolarization (Ogita et al., 1995; ZaÈngerle et al., 1992). They suggest that GSH originates from neuronal compartment, since this release is Ca2+-dependent, and that it may modulate neuronal excitation through interaction with receptors for excitatory aminoacids. The percentage of GSH binding measured at the equilibrium point may con®rm the presence of binding sites for GSH in synaptosomal membrane receptors, as also reported by others (Ogita and Yoneda, 1989; Guo et al., 1992). These data may indicate an involvement of this transporter in the role suggested for GSH in synaptic neurotransmission, (Guo et al., 1992). An other possible physiological role of GSH synaptosomal transporter may be to supply neurons with GSH. Moreover, this transporter, together with those found by Kannan et al. (1996) in bovine brain capillary and by Sagara et al. (1996) in astroglial cells, may contribute to maintaining a GSH interorgan cycle as well as GSH homeostasis. Recently it has been suggested that neuronal GSH metabolism is linked to that in glial cells. GSH levels in astrocytes are higher than those measured in neurons (Sagara et
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al., 1996; Dringen et al., 1997; Juurlink et al., 1996) and it has been demonstrated that GSH is released from astrocites by mediated transport, and that stress oxidative increases GSH eux (Sagara et al., 1996). Increases of GSH extracellular levels in the brain have also been measured by microdyalisis experiments in ischemic rat striatum (Orwar et al., 1994). Therefore it has been suggested that the release of GSH from astroglial cells may contribute to protect neurons against oxidative injury and to the maintenance of the GSH neuronal pool by the gGT breakdown of extracellular GSH, subsequent uptake of its constituent aminoacids and intracellular GSH resynthesis (Sagara et al., 1996; Dringen et al., 1997). These authors exclude a direct GSH uptake into neurons, but no experimental data are reported. Moreover, we do not detect g-GT activity in synaptosomes. The kinetic parameter values of GSH eux from astrocytes (Km 36 mM and Vmax 0.39 nmol/ min/mg protein) are very dierent from those measured for GSH uptake in SMV, indicating the presence of two dierent types of GSH transporters, which probably correspond to dierent physiological roles. The high uptake rate and anity for GSH of synaptosomal transporter suggest that astrocytes may eectively supply neurons with GSH. This would be important, considering that GSH is involved in regulating levels of neuroactive aminoacids and that low levels of GSH and of g-glutamylcysteine synthetase expression have been found in cerebral cortex (Kang et al., 1997). Changes in GSH synthesis and a decrease in GSH content in cerebral cortex have been related to susceptibility to neuropathologies often associated with oxidative stress (Cassarino et al., 1997). Acknowledgements This work was supported by the Italian Association for Cancer Research (AIRC) and by the Ministero della Pubblica Istruzione e della Ricerca Scienti®ca e Tecnologica (40%). References AragoÂn, M.C., AgulloÂ, L., GimeÂnez, C., 1988. Depolarizationinduced release of glycine and D-alanine from plasma membrane vesicles derived from rat brain synaptosomes. Biochimica Biophysica Acta 941, 209±216. Bains, J.S., Shaw, C.A., 1997. Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Research Reviews 25, 335±358. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248±254. Cassarino, D.S., Fall, C.P., Swerdlow, R.H., Smith, T.S., Halvorsen, E.M., Miller, S.W., Parks, J.P., Parker Jr, W.D., Bennett Jr, J.P.,
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